The purpose of this project is to understand the role that non-histone proteins play in chromosome structure and condensation. Over the past eight years our efforts have focussed on protein components of the chromosome scaffold fraction. While the precise biological meaning of the scaffold fraction is a subject of ongoing debate, our results suggest that it may contain a number of proteins that have bona fide roles in chromosome structure. In previous experiments funded by this project, we identified the major protein of the scaffold fraction as DNA topoisomerase II. During the upcoming grant period, we will follow two major avenues of research. In the firsT, we will characterize three new scaffold proteins: INCENPs, ScII and ScIII. A new assay will be used to determine if they are tightly associated with chromosomal structures in vivo. Unlike topo II, these are not previously known proteins, and we will thus characterize them in detail at the molecular level by cloning and sequencing their cDNAs. We will also map their distribution in chromosomes by confocal and electron microscopy. Preliminary observations suggest that the binding affinities of the INCENPs change at the metaphase:anaphase transition, and we will therefore study their modification status and stability across the cell cycle. Since the INCENPs, ScII and ScIIl are new proteins, we have no clues as to their function in the chromosome. We will therefore undertake to determine this in vivo using antibody microinjection and transient transfection with partial cDNAs encoding putative isolated protein domains. In addition to these functional studies, we will use our antibody libraries to attempt to identify homologues of these proteins in Drosophila for future collaborative genetic analysis. In vitro studies that complement the in vivo studies are also proposed. We have succeeded in developing a new extract system from somatic cells that causes interphase nuclei to undergo chromosome condensation in vitro. The unique aspect of this system is that unlike frog egg extracts that have similar activity, somatic cell extracts lack large stockpiles of structural proteins. They can thus be used to assay the role of structural proteins in chromosome condensation. Where possible, we will take advantage of our observation that certain of the major chromosomal non-histone proteins such as topoisomerase II are selectively lost during chicken erythropoiesis. We will use late erythroblast nuclei as substrates in mitotic extracts from which specific non-histone proteins have been depleted by immunoprecipitation. This provides a powerful assay for analysis of the role of individual non-histones in chromosome condensation. The molecular and structural characterization of these proteins coupled with our powerful new in vitro assay system should enable us to begin to assign functions to some members of this largely uncharacterized group of chromosomal proteins.